Rosaramicin (1), a 16-membered ring macrolide antibiotic, was isolated from the culture filtrate of Micromonospora rosaria'). The biosynthesis of rosaramicin was previously investigated3,3) and established the incorporation of radiolabeled acetic, propionic, and butyric acids and L-methionine into rosaramicin. However, we wished to explore the biosynthetic route of rosaramicin using intermediates and idiotrophs. Therefore, mutants blocked in rosaramicin biosynthesis were generated by the treatment of the rosaramicin producing strain M. rosaria (SCC 957, ATCC 29337) with NTG. Mutant T6071 was obtained which produced a mixture of two 16-membered lactones, 20-deoxorosaranolide (2) and 20-deoxo-12,13 -desepoxy -12,13 -dehydrorosaranolide (3). This paper describes the fermentation, isolation, and characterization of the two components.The aglycone complex, initially detected in test tube and 250 ml Erlenmeyer flask cultures, was produced in a 150-liter New Brunswick Scientific Fermentor containing 100 liters of a medium consisting of 1.5%. Amberex-1003, 0.3% fish peptone, 5 % potato dextrin, 0.2 % Pharmamedia (cotton seed flour), 1 % maltose, 0.02 % sodium thiosulfate, 0.001% cobalt chloride, 0.001 % manganese chloride, 0.1%. SAG-471 antifoam, and tap water. The pH of the medium was adjusted to 6.8 prior to sterilization. After inoculation, the culture was incubated at 30°C under aeration (57 liters/minute) with agitation (300 rpm) for 66 hours.The harvested fermentation broth (100 liters) was adjusted to pH 8.0 and extracted twice with 200-liter volumes of ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure to give a crude residue. The residue was dissolved in 95 % aqueous ethanol and purified further on a column of Sephadex LH-20 (1,500 ml, Pharmacia Co.) using 95 % aqueous ethanol as the eluant at a flow rate of 1 ml/minute. Elution of the aglycone mixture occurred between 2 and 3 liters of eluant. Fractions were tested for the aglycone complex by thin-layer chromatography using Whatman 0.25 mm LK6DF silica gel plates developed for one hour in a solvent system of chloroform -methanol (9: 1, v/v) followed by treatment with sulfuric acid. Those fractions which contained compound with an Rf 0.7 were combined and concentrated to dryness. The residue was dissolved in a small volume of 95 % aqueous ethanol, water added, and the solution was lyophilized to yield 16 g of dry powder containing the rosaramicin aglycone complex.The complex was shown to be a mixture of components 2 and 3 in the ratio of 1: 4 based upon PMR, CMR, and MS data. Further purification by high performance liquid chromatography led to the separation of the complex into components 2 and 3 with greater than 95 purity. The complex was injected onto an E. S. Industries Chromegabond C-8 (10 it), 500 mm x 9.6 mm column and eluted with an acetonitrile -0.01 M ammonium acetate buffer (pH 4.0) (4: 6) mobile phase. Each injection of 80 mg of mixture yielded 13.8 mg of 2 and 42.2 mg of 3.The molecular formula of 2 was...
